Help: Possible ligation of multiple plasmids when only self-ligation is wanted

I need help in distinguishing a self-ligated vector and vectors ligated to each other!

Basically the vector was double-digested with two enzymes (NotI to create the ends, XhoI to cut up the fragment in between) to remove a section of the plasmid, then it's treated with T4 to religate back. But then the horror struck me that I could not differentia between a self-ligated vector, and two vectors that ligate to each other with restriction enzymes!

A PCR reaction that flanks the cut site confirms that the ligation is in the correct orientation, but I'm just not sure whether it is amplified from a single site, as is the case in a self-ligated vector, or whether it is coming from two sites at the two points where the two vectors ligate. I thought about quantifying the PCR product since the double-plasmid should give more product given that it has two templates sites, but I'm not sure how to standardise the amount of DNA template as one double plasmid is going to weigh as much as TWO self-ligated plasmid, and so it's all back to square one. Also I'm not sure if running the different plasmids on gel would help; is there any way to run and view these vectors on the gel, then convincingly know which is a double-plasmid which is not? The vector is pretty big (18+kb) so a double-plasmid would be like ~37kb.

Can anyone offer any suggestion or help? Thank you so much in advance!

2. It is rare for normal colE1 plasmid origins to function when there is more than one on a single plasmid. This is not necessarily true for other origins, such as p15, or pSC101.

I have only had a problem with this once, when an origin mutated to be inactive, and was present along with an unmutated one in a single plasmid. This resulted in an apparent single base in sequencing results which had two different base IDs. No amount of restreaking for single colonies eliminated it, but cutting the vector and religating it did solve the issue. These could occur more often than we know, but perhaps it makes little difference. Your uncut plasmid gel should show a difference.

You can minimize the number of these produced by lowering the concentration of vector in your ligation reactions.

I have only had a problem with this once, when an origin mutated to be inactive, and was present along with an unmutated one in a single plasmid. This resulted in an apparent single base in sequencing results which had two different base IDs.

Wow. Never seen that happen before. That event must have been weird.

May your PCR products be long, your protocols short and your boss on holiday

2. It is rare for normal colE1 plasmid origins to function when there is more than one on a single plasmid. This is not necessarily true for other origins, such as p15, or pSC101.

I have only had a problem with this once, when an origin mutated to be inactive, and was present along with an unmutated one in a single plasmid. This resulted in an apparent single base in sequencing results which had two different base IDs. No amount of restreaking for single colonies eliminated it, but cutting the vector and religating it did solve the issue. These could occur more often than we know, but perhaps it makes little difference. Your uncut plasmid gel should show a difference.

You can minimize the number of these produced by lowering the concentration of vector in your ligation reactions.

Thanks so much for the suggestion; I'm gonna try cutting then religating the vector in low concentration. The vector is going to be used for subsequent insertional recombination so I wanted to get the vector right before i put in anything extra.

Plasmid concatemers could result in any cloning reaction, whether there's insert present or not. The reasons they're not a problem is that such a construct is disfavored for many reasons -- phage434 points one out, above -- but there are others, like copy control enzymes interfering with one another and other gene dosage effects, plasmid-encoded resolvases, and that the larger plasmid is less likely to transform competent cells, and thus is selected against. You can minimize the possibility further using proper vector:insert ratios as well.

In fact, even without a ligation reaction involved, plasmid concatemers could occur in any cell carrying a plasmid with a copy number greater than one by homologous recombination.

But, though such a construct can theoretically form, the fact that this is so rarely a problem testifies empirically that you needn't worry about it. When you add your insert in, there's a much greater chance of recovering a clone with concatenated inserts, but even this is a rare event.

Plasmid concatemers could result in any cloning reaction, whether there's insert present or not. The reasons they're not a problem is that such a construct is disfavored for many reasons -- phage434 points one out, above -- but there are others, like copy control enzymes interfering with one another and other gene dosage effects, plasmid-encoded resolvases, and that the larger plasmid is less likely to transform competent cells, and thus is selected against. You can minimize the possibility further using proper vector:insert ratios as well.

In fact, even without a ligation reaction involved, plasmid concatemers could occur in any cell carrying a plasmid with a copy number greater than one by homologous recombination.

But, though such a construct can theoretically form, the fact that this is so rarely a problem testifies empirically that you needn't worry about it. When you add your insert in, there's a much greater chance of recovering a clone with concatenated inserts, but even this is a rare event.

But a it's good question, klwenk -- it shows you're thinking...!

Thanks alot for the insights! My colleagues raised the issue and we wanted to be sure...so we are hoping to come up with a diagnostic assay that can differentiate the two with certainty. But since PCR and RE assays dont seem like they would work, we are left with running the uncut clones and see if any weird ones come up. But if like you said the event is a very rare one, we might probably end up with identical patterns for all of them afterall!